title: Genome-wide annotation and quantitation of translation by ribosome profiling.
abstract: Recent studies highlight the importance of translational control in determining protein abundance, underscoring the value of measuring gene expression at the level of translation. A protocol for genome-wide, quantitative analysis of in vivo translation by deep sequencing is presented here. This ribosome-profiling approach maps the exact positions of ribosomes on transcripts by nuclease footprinting. The nuclease-protected mRNA fragments are converted into a DNA library suitable for deep sequencing using a strategy that minimizes bias. The abundance of different footprint fragments in deep sequencing data reports on the amount of translation of a gene. Additionally, footprints reveal the exact regions of the transcriptome that are translated. To better define translated reading frames, an adaptation that reveals the sites of translation initiation by pre-treating cells with harringtonine to immobilize initiating ribosomes is described. The protocol described requires 5 to 7 days to generate a completed ribosome profiling sequencing library. Sequencing and data analysis requires an additional 4 to 5 days.
authors.affiliation: Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, California.|California Institute for Quantitative Biosciences, San Francisco, California.|Department of Embryology, Carnegie Institution for Science, Baltimore, Maryland.
authors.forename: Nicholas T
authors.initials: NT
authors.lastname: Ingolia
authors.affiliation: Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, California.|California Institute for Quantitative Biosciences, San Francisco, California.
authors.forename: Gloria A
authors.initials: GA
authors.lastname: Brar
authors.affiliation: Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, California.|California Institute for Quantitative Biosciences, San Francisco, California.
authors.forename: Silvia
authors.initials: S
authors.lastname: Rouskin
authors.affiliation: Department of Embryology, Carnegie Institution for Science, Baltimore, Maryland.|Department of Biology, The Johns Hopkins University, Baltimore, Maryland.
authors.forename: Anna M
authors.initials: AM
authors.lastname: McGeachy
authors.affiliation: Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, California.|California Institute for Quantitative Biosciences, San Francisco, California.
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Weissman
mesh_terms: D019295:Computational Biology / Q000379:methods; D020869:Gene Expression Profiling / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D014176:Protein Biosynthesis; D012270:Ribosomes / Q000378:metabolism*; D013997:Time Factors